Effect of ligand on GR LBD expression. The red arrow denotes GR LBD expressed with seven different ligands. In some cases, the addition of a methyl group to the ligand could make the difference in increased expression levels.

Effect of the C808S mutation and ligand type on MR expression. Although the C808S mutation is equivalent to the GR F602S mutation, it has a more pronounced effect on GST-MR LBD expression. This mutation produced increased expression of MR in the presence of a variety of ligands (L1, ligand 1; L2, ligand 2; L3, ligand 3). High levels of protein expression still require the presence of ligand during cell growth.

Figure 6

Mineralocorticoid receptor. The MR C808S mutant expressed well in the presence of potent compounds and its structure has been determined in complex with several ligands (Bledsoe et al., 2005 ▶).

Progesterone receptor LBD. Cocrystals of the ligand of interest were obtained when the protein was initially expressed with a lower affinity ligand. The ligand of interest was then added in molar excess and included throughout protein purification.

Pure estrogen receptor α was obtained when the cells were lysed in the presence of urea and subsequently purified by estradiol affinity chromatography. The key to obtaining well behaved protein was refolding this protein in the presence of a ligand. This gave sufficient quantities of soluble protein without special growth conditions, i.e.inclusion of the ligand during protein expression (Brzozowski et al., 1997 ▶; Tanenbaum et al., 1998 ▶).

Work is still in progress on kinase 2, which copurifies with HSP90 on all chromatography mediatested to date. We were not able to disrupt the kinase 2–HSP90 complex by the addition of a variety of salts, detergents or ATP. However, once a high-affinity ligand was included during the early steps of the purification, we obtained soluble kinase 2 without the HSP90. Although we do not yet have crystals of this protein, we now have monomeric protein to use for crystallization trials.

Initial crystals of the viral protein–ligand complex in Fig. 11 ▶showed no diffraction. However, when this protein complex was heated to 310 K for 5–10 min, followed by incubation on ice and centrifugation with a 0.2 µm filter, the resulting crystals diffracted to 2.8 Å (Wang & Nolte, 2006 ▶). The addition of 0.1% β-octylglucoside to the protein solution was key to obtaining crystals of some of these viral protein–ligand complexes.

Figure 11

Crystals of a heat-treated viral protein–ligand complex diffract to 2.8 Å. The addition of 0.1% β-octylglucoside to the protein solution was required to grow crystals of some of these complexes.

Protein concentration/ligand concentration

Sometimes it is possible to concentrate our protein and then add the ligand to form the complex. However, if the ligand is insoluble, it may cause the protein to precipitate when it is at higher concentrations. It may be necessary to add dilute ligand to diluted protein to achievegood ligand binding with these very insoluble compounds. Kinase 4 had to be diluted to 1 mg ml−1 and then complexed with dilute ligand at a 1:3 protein:ligand ratio to achieve a stable complex that yielded well diffracting crystals (Fig. 12 ▶). The majority of these complex cocrystals were grown by cross-seeding usingapo crystals.

Figure 12

A protein concentration of 1 mg ml−1 during complex formation and cross-seeding were critical to obtaining cocrystals of the kinase 4 complexes.

Examples have been presented showing the effects of temperature, protein concentration, the use of additives and ligand concentration on the formation of protein–ligand complexes with insoluble compounds. However, there are additional ways to tackle the problem of insoluble ligands: homogenize the powdered with a small pestle, mix the ligand with tinybeads and vortex or sonicate to homogenize the powder and soak the crystal in cryoprotectant first before adding the ligand.

Ligand exchange prior to cocrystallization

What if the protein already has a natural ligand in the ligand-binding pocket (Fig. 13 ▶)? How do we obtain cocrystals of a protein–inhibitor complex?

Cross-seeding

Apo crystals of kinase 5 were easily reproduced, but growing crystals of the ligand complexes proveddifficult. Apo crystals were used for cross-seeding into Hampton Research Crystal Screen to find initial crystallization conditions for the ligand complexes. Crystal Screen reagent 28 gave the best results and was optimized. All of the subsequent cocrystals were grown by cross-seeding with either apo crystals or crystals of other ligand complexes into this reagent (Fig. 15 ▶).

Figure 15

Crystals of the kinase 5 ligand complexes were obtained by cross-seeding.

Real time

Some proteins require the presence of a ligand during expression to obtain sufficient quantities of stable protein for crystallization trials. In some cases, the ligand used during protein expression is not the ligand of choice for structural studies. In RTISCC, the ligand of interest is added to the crystallization drop to competeout the first ligand used during expression (Fig. 16 ▶).

Figure 16

Real-time in situ competition crystallization (RTISCC). The ligand of interest competes out the original ligand used during protein expression.

Soaking ligands into existing crystals

Soaking crystals with ligands is often the method of choice to obtain crystals of protein–ligand complexes owing to the ease of the method. However, there are several factors to consider. The crystals may be fragile and soaking in a stabilization buffer or cross-linking may be required. The soaking time and inhibitor concentration need to be optimized, as many protein crystals are sensitive to the solvents used to dissolve the ligands. Additives may be required to achieve effective ligand binding during the soak time and/or during the subsequent cryoprotectant exchange. Finally, you may have cocrystals of one ligand complex and need to exchange the original ligand with a different ligand (replacement soaking; Skarzynski & Thorpe, 2006 ▶). The FASTfragment-based screening developed by Structural Genomix Pharmaceuticals is an example of a high-throughput soaking-type system that has been quite successful (Burley, 2004 ▶).

Although soaking ligands into crystals may be the method of choice for a particular protein, it is preferable to validate the soaking system with cocrystallization experiments when possible. The fullrange of conformational changes may not be seen in instances where the ligand has been soaked into the crystal. However, in the case of cyclin A–cdk2 crystals, the cyclin A restrictsmovement of the cdk2 and soaking in this system is a valid approach.

Stabilization of crystals/use of an additive during the soak

Crystals are often put into ‘stabilization’ buffers before they are immersed in the ligand solution. These buffers may contain increased concentrations of the precipitant(s) and a stepwise/gradual increase in reagent concentration or the introduction of a cryoprotectant may be required so that the crystals are not damaged.

Cross-linking with glutaraldehyde and the use of PEG 400 with large ligands improved the success rate for soaks in the cyclinA–cdk2 system.

Although PEG 400 has been useful for soaking large ligands into the cyclinA–cdk2 crystals, there are a variety of reagents that have proved useful in other systems, including Jeffamines, sugars, methylpentanediol (MPD) and a variety of PEGS. We often use some of the reagents in the limited additive screen in Table 1 ▶ to improve ligand solubility. This can be accomplished in several ways. The additive may be added directly to the precipitating reagent in the well and thoroughly mixed. Subsequently, the ligand is added to a drop of this additive/precipitating reagent mixture in a 1:1 ratio and then added to the protein drop. Alternatively, the ligand may be mixed with the additive and then added to the protein drop. The exactratios of additive:ligand:precipitating reagent need to be optimized, as this can vary greatly depending on the type of ligand.

In protease 1, cocrystallizations yielded very few protein–ligand complexes, so a soaking strategy was devised. Since the apo crystals were rather fragile and the ligands were quite insoluble, an additive was needed that stabilized the crystal and improved the ligand solubility. Xylitol was added to the precipitating solution to a final concentration of ∼2–5%. 1 µl of this mixture was added to the protein drop to stabilize the crystals (∼15–60 min). Next, the inhibitor was added to the additive plus precipitating reagent mix (∼2–5 µl ligand plus 500 µl precipitating reagent). 1 µl of this ligand mixture was then added to the crystals (Fig. 18 ▶). This procedure was the only method that led to solution of structures of the protease–ligand complexes.

Figure 18

Protease 1. The use of xylitol during the soaking stabilized the crystal and improved the solubility of the ligands. If the ligand was not mixed in the xylitol plus precipitating mixture, the efficiency of obtaining protein–ligand complexes greatly decreased.

Soaking time/ligand concentration

The previous example showed crystals of cyclinA–cdk2 that were sensitive to handling where cross-linking and the use of an additive were critical for successful ligand soaks. However, in cdk2, a second approach of diluting the inhibitor and using longer incubation times worked well in obtaining inhibitor complexes. 1–3 µl of a 50 mMstock inhibitor solution was added to 500 µl well reservoir. 1 µl of this diluted ligand was then added to a 4–5 µl drop containing the cdk2 crystals (Fig. 19 ▶). Incubation times ranged from several days to 2–3 months.

Figure 19

Inhibitor soaks of cdk2 gave crystals that diffracted to ∼2 Å. Dilute inhibitor concentrations coupled with long incubation times gave the best complexes.

Ligand exchange in crystals

There are instances where it is easy to grow crystals that contain a natural ligand or cocrystals with one inhibitor, but not with a new ligand or template. In this case, the new ligand of interest may be soaked into the existing cocrystals, substituting it for the original compound. When performing this ‘replacement soaking’ (Skarzynski & Thorpe, 2006 ▶), one must consider the binding constant of the new ligand. It may be difficult to introduce a new lower affinity ligand into the system and substitute it for a much higher affinity ligand that is already bound to the protein. The success of the ligand replacement can be seen when the electron-densitymap is calculated.

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Conclusions

There are a wide variety of techniques available to the investigator for obtaining protein–ligand complexes. These include adding ligands during protein expression to obtain soluble protein, addition of the ligand during protein purification, cocrystallization of the ligand with the purified protein and soaking ligands into crystals. It is reasonable to start by soaking the ligands into crystals as this is the easiest method or to try cocrystallizing the ligand with the purified protein. If these are not successful, further optimization of the protein expression and purification may be necessary.

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